The Olivocerebellar Projection Mediates Ibogaine-Induced … · 1997-11-04 · Purkinje cells have...

The Olivocerebellar Projection Mediates Ibogaine-InducedDegeneration of Purkinje Cells: A Model of Indirect,Trans-Synaptic Excitotoxicity

Elizabeth O’Hearn and Mark E. Molliver

Departments of Neuroscience and Neurology, The Johns Hopkins University School of Medicine,Baltimore, Maryland 21205

Ibogaine, an indole alkaloid that causes hallucinations, tremor, andataxia, produces cerebellar neurotoxicity in rats, manifested bydegeneration of Purkinje cells aligned in narrow parasagittal bandsthat are coextensive with activated glial cells. Harmaline, a closelyrelated alkaloid that excites inferior olivary neurons, causes thesame pattern of Purkinje cell degeneration, providing a clue to themechanism of toxicity. We have proposed that ibogaine, like har-maline, excites neurons in the inferior olive, leading to sustainedrelease of glutamate at climbing fiber synapses on Purkinje cells.The objective of this study was to test the hypothesis that in-creased climbing fiber activity induced by ibogaine mediates ex-citotoxic Purkinje cell degeneration. The inferior olive was phar-macologically ablated in rats by a neurotoxic drug regimen using3-acetylpyridine, and cerebellar damage attributed to subsequentadministration of ibogaine was analyzed using immunocytochem-

ical markers for neurons and glial cells. The results show thatibogaine administered after inferior olive ablation produced little orno Purkinje cell degeneration or glial activation. That a lesion of theinferior olive almost completely prevents the neurotoxicity dem-onstrates that ibogaine is not directly toxic to Purkinje cells, butthat the toxicity is indirect and dependent on integrity of theolivocerebellar projection. We postulate that ibogaine-induced ac-tivation of inferior olivary neurons leads to release of glutamatesimultaneously at hundreds of climbing fiber terminals distributedwidely over the surface of each Purkinje cell. The unique circuitryof the olivocerebellar projection provides this system with maxi-mum synaptic security, a feature that confers on Purkinje cells ahigh degree of vulnerability to excitotoxic injury.

Key words: ibogaine; harmaline; Purkinje cell; cerebellum;excitotoxicity; climbing fiber; inferior olivary nucleus; microglia

Ibogaine is a psychoactive five-ringed indole alkaloid extractedfrom the root of an African plant, Tabernanthe iboga (Dhahir,1971). This drug is a CNS stimulant with multiple pharmacolog-ical effects that include production of hallucinations, tremor, andataxia (Schneider and Sigg, 1956; Popik et al., 1995). Based onanecdotal reports that ibogaine may suppress craving associatedwith drug addiction and may reduce signs of drug withdrawal inhumans (Lotsof, 1985, 1986, 1995; Sheppard, 1994), this com-pound has been considered for clinical use in the treatment ofdrug addiction. However, based on a study to detect neurotoxiceffects of ibogaine, this laboratory reported that treatment witheither ibogaine or the related drug harmaline can lead to neuronalinjury in the cerebellum of rats. The cytotoxic effects are locatedpredominantly in the vermis of the cerebellum and manifested byneuronal degeneration accompanied by marked gliosis (O’Hearnand Molliver, 1993; O’Hearn et al., 1993). The neurotoxicityinduced by ibogaine is selective for Purkinje cells and is charac-terized by a distinctive spatial pattern, such that degeneratingPurkinje cells are aligned in narrow longitudinal bands within thevermis and, less frequently, in the paravermis or hemispheres.Activated microglia and astrocytes form sagittally oriented radialstripes that are in register with the longitudinal bands in whichPurkinje cells have degenerated (Fig. 1).

Administration of ibogaine produces abnormal motor behaviorthat includes a high frequency tremor associated with markedataxia in mice (Zetler et al., 1972; Singbartl et al., 1973) and inrats (Glick et al., 1992; O’Hearn and Molliver, 1993). The behav-ioral effects induced by ibogaine are indistinguishable from ef-fects produced by two related indole alkaloids, harmaline andibogaline (Singbartl et al., 1973; Zetler et al., 1974). The lattertwo drugs have powerful CNS excitatory effects manifested by amarkedly increased firing rate of neurons within the inferiorolivary nucleus (De Montigny and Lamarre, 1973, 1974; Llinasand Volkind, 1973). Based on the pharmacological similaritiesamong these three drugs, we propose that ibogaine, like harma-line and ibogaline, is likely to increase the excitability and firingof neurons in the inferior olive. The mechanisms by which ibo-gaine and related b-carboline compounds produce increased in-ferior olive activity are not fully characterized, and other sites atwhich ibogaine may act in the CNS have not yet been clearlyidentified. Based on the evidence available, we have postulatedthat the degeneration of Purkinje cells produced by both ibogaineand harmaline results from excitotoxic injury (O’Hearn and Mol-liver, 1993; O’Hearn et al., 1995). In accord with the excitotoxichypothesis (Olney, 1978; Choi, 1988), these drugs should producea sustained increase in neuronal firing in the inferior olive,leading to release of excessive glutamate from climbing fiberterminals that synapse on longitudinal arrays of Purkinje cells.The repetitive release of an excitatory neurotransmitter, sus-tained over many hours, is likely to produce irreversible, excito-toxic damage to Purkinje cells, followed by their degeneration.

The purpose of the present study is to test the hypothesis thatibogaine-induced degeneration of Purkinje cells results from ex-

Received April 30, 1997; revised Sept. 3, 1997; accepted Sept. 8, 1997.This work was supported by United States Public Health Service Grants DA

08692, DA 00225, and NO1DA-3-7301.Address all correspondence to Dr. Elizabeth O’Hearn, Department of Neuro-

science, PCTB 1032, Johns Hopkins University School of Medicine, 725 NorthWolfe Street, Baltimore, MD 21205.Copyright © 1997 Society for Neuroscience 0270-6474/97/178828-14$05.00/0

The Journal of Neuroscience, November 15, 1997, 17(22):8828–8841

cessive and prolonged activation of the olivocerebellar projection.To evaluate the role of climbing fibers in mediating neuronaldegeneration attributed to ibogaine, the inferior olivary nucleuswas chemically ablated in rats by means of a systemic, neurotoxicdrug regimen that has been shown to produce relatively selectivedegeneration of neurons in the inferior olive (Llinas et al., 1975;Anderson and Flumerfelt, 1980; Balaban, 1985). Six days aftersustaining olivary lesions, rats were injected with ibogaine andallowed to survive for an additional week. The animals werekilled 1 week after ibogaine treatment, and brain sections wereobtained to analyze the degree of Purkinje cell degeneration andto verify inferior olive ablation. The extent of Purkinje celldegeneration produced by ibogaine after ablation of the inferiorolive was compared with ibogaine-induced degeneration in nor-mal rats with intact inferior olives. If Purkinje cell degenerationinduced by ibogaine is mediated via the olivocerebellar projec-tion, then ablation of the inferior olive before ibogaine adminis-tration would be expected to prevent the neuronal loss.

MATERIALS AND METHODSSubjects. Male Sprague Dawley rats (n 5 40, 175–220 gm; HarlanSprague Dawley, Indianapolis, IN) were housed individually on the daybefore and the day after drug treatments, after which they were housedin groups of three or four. No solid food was available from the nightbefore and up until drug administration, after which they had free accessto food (Prolab RMH 1000; PMI Feeds, St. Louis, MO) and water.Cages were located in a temperature-regulated room (70°F) with a 12 hrlight /dark cycle.

All solutions were administered by intraperitoneal injection. Drugdoses are expressed as weights of the salt (ibogaine-HCl; obtained fromthe National Institute on Drug Abuse) or of the free base 3-acetylpyridine (3-AP), harmaline, or nicotinamide (Sigma, St. Louis,MO) per kilogram of rat body weight. Solution concentrations were asfollows: 3-AP, 25 mg/ml saline; harmaline, 7 mg/ml saline; nicotinamide,60 mg/ml saline; and ibogaine-HCl, 10 mg/ml distilled water. Controlrats received one injection of normal saline in a volume equal to that ofibogaine administered to treated rats.

Treatment paradigms. Animals were assigned to four treatment groups:(1) group A, the 3-AP regimen: 3-AP (68–70 mg/kg) followed byharmaline (15–20 mg/kg) 2 hr later and by nicotinamide (300 mg/kg) anadditional 2.5 hr later (4.5 hr after 3-AP); after 6 d these rats were givenibogaine (100 mg/kg) and observed for 1 week before killing and neu-roanatomic examination (n 5 17); (2) group B, 3-AP, harmaline, andnicotinamide as in group A, followed 6 d later by saline (n 5 8); (3) groupC, ibogaine alone (100 mg/kg) on the same day it was administered toanimals in group A (n 5 6); and (4) group D, saline (n 5 6) or notreatment (n 5 3) on the same day ibogaine was given to animals ingroups A and C.

This study was conducted in four sequential trials, each of whichincluded animals from the four treatment groups. In proceeding throughthe four trials, the dose of 3-AP was varied (68–70 mg/kg), as washarmaline (15–20 mg/kg) to maximize selective neuronal loss in theinferior olive. Harmaline was administered 2 hr after 3-AP (instead of 3hr, as used by Llinas et al., 1975) to increase the selectivity of thisregimen for inferior olivary neurons. Based on reports that 1–6 d arerequired for ablation of neurons of the inferior olive after the 3-APregimen (Desclin and Escubi, 1974; Llinas et al., 1975; Sotelo et al.,1975), ibogaine was given 6 d after the 3-AP regimen.

Immunocytochemical evaluation. One week after ibogaine (or saline)treatment, animals were deeply anesthetized with pentobarbital (80mg/kg, i.p.) and perfused through the left ventricle with cold 4% para-formaldehyde in 0.15 M phosphate buffer, pH 7.4. Brains were post-fixedin the same solution for 4–6 hr and cryoprotected in 10% DMSO in PBS.The cerebellum was sectioned at 40 mm on a freezing sliding microtome.Every fourth section was collected and prepared for immunohistochem-ical examination or Nissl stain.

Immunohistochemical staining was performed on freely floating sec-tions. Primary antibodies included neuronal markers for calbindin (1:8000, R17; a gift from P. Emson, Cambridge University) and calcium-and calmodulin-dependent protein kinase II (Cam-kin II) (1:1000;Boehringer Mannheim, Indianapolis, IN). Microglial markers used were

MRC OX42 (1:1000; Serotec, Oxford, UK), an antibody to the comple-ment receptor 3, and MRC OX6 (1:3000; Serotec), an antibody to themajor histocompatibility complex II (MHC II) antigen. Astrocytes werelabeled with an antibody to glial fibrillary acidic protein (GFAP) (1:15,000; Dako, Via Real, CA). Primary antibody incubation solutionscontained 0.2% Triton X-100 and 2% normal goat or horse serum(Vector Laboratories, Burlingame, CA) in PBS or Blotto. To reducebackground staining for Cam-kin II, OX42, and OX6, sections wereimmersed in solutions containing 0.2% Triton X-100, 2% normal horseserum, and Blotto for 60 min before incubation in gently agitated primaryantibody solutions for 48–72 hr at 4°F. Primary antibodies were visual-ized using Vectastain ABC Elite reagents (Vector) and the chromagen3,39-diaminobenzidine (Sigma). Sections were examined with a LeitzDMRB/E microscope equipped for bright field and differential interfer-ence contrast.

RESULTSMotor effects of drug treatmentIbogaine aloneAfter receiving ibogaine (100 mg/kg, i.p., once), rats displayed apredictable sequence of behavioral signs. Within 3 min, theydeveloped a high frequency tremor of the trunk, head, and limbs,followed by marked ataxia manifested by a wobbling gait andfrequent falls. During the initial 5 min many rats exhibited myo-clonic jerks of the limbs, followed by brief episodes (5–10 sec) thatincluded extension of the head, rapid patting movements offorelimbs with extension of digits, and repetitive facial move-ments. These seizure-like episodes started and stopped abruptlyand were typically followed by immobility for several seconds,after which ataxia returned. Not uncommonly, rats were pro-jected off the floor by extensor limb movements during the first 5min after ibogaine treatment.

The initial episode of ibogaine-induced tremor, ataxia, andmyoclonus was followed by a period of marked hypotonia. At8–10 min after receiving ibogaine, rats lay prone with their headson the floor and with limp, motionless limbs, yet they remainedawake with eyes open. The only visible spontaneous movementswere those of abdominal and thoracic muscles related to respira-tion. Although they were extremely hypotonic, the rats respondedto noxious stimuli; a foot pinch elicited withdrawal of limbs. Notremor was present during the hypotonic stage, which lasted for15 min and was followed by gradual return of truncal tone andspontaneous limb movements. Recovery of muscle tone and up-right posture at 30–60 min after treatment were accompanied byrecurrence of tremor and ataxia. The latter signs diminished over18 hr and, at 24 hr after treatment, the behavior of treated ratscould not be distinguished from that of controls. The initialtremor and the tremor after the hypotonic period were identicaland were estimated to have a frequency of 8–10 Hz. The tremorcaused by ibogaine was indistinguishable from that induced byharmaline.

3-AP, harmaline, and nicotinamide regimenAfter administration of 3-AP alone, rats displayed no abnormalmotor signs acutely. Injection of harmaline, as part of the 3-APregimen, rapidly led, within minutes, to a high-frequency gener-alized tremor and ataxic gait, followed by transient hypotonia,similar to the motor effects produced by ibogaine. The high-frequency tremor gradually disappeared after ;12 hr. From thefollowing day until the time of killing, the 3-AP-treated ratsexhibited persistently abnormal neurological signs, as describedpreviously (Llinas et al., 1975; Balaban, 1985) and that were notpresent after ibogaine alone. They became profoundly ataxic andhad an unusual steppage gate, described as “mud-walking” (Lli-

O’Hearn and Molliver • Climbing Fibers Mediate Purkinje Cell Degeneration J. Neurosci., November 15, 1997, 17(22):8828–8841 8829

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Figure 1. Ibogaine causes degeneration of Purkinje cells and activation of microglia in discrete radial bands of cerebellar cortex. A, B, Purkinje cellsof cerebellar vermis at low ( A) and high ( B) magnification 7 d after receiving ibogaine (100 mg/kg once). Unstained gaps in the Purkinje cell andmolecular layers indicate regions in which Purkinje cells have degenerated (Cam-kin II immunoreactivity, coronal sections). C, D, Clusters of activatedmicroglial cells form darkly stained radial stripes within the cerebellar vermis, in sections adjacent to those showing Purkinje cells. The stripes containingactivated microglia are approximately coextensive with regions of Purkinje cell loss (compare densely stained stripes in C (Figure legend continues)

8830 J. Neurosci., November 15, 1997, 17(22):8828–8841 O’Hearn and Molliver • Climbing Fibers Mediate Purkinje Cell Degeneration

nas et al., 1975). Starting 24 hr after treatment with the 3-APregimen, a brief tremor was observed during the initiation of heador trunk movements. These 3-AP-treated rats differed from thosethat received ibogaine alone, because the latter displayed notremor or ataxia beyond 24 hr after treatment.

Ibogaine in 3-AP-, harmaline-, andnicotinamide-pretreated animalsRats that received ibogaine 6-d after the 3-AP regimen exhibitedmyoclonic jerks, brief seizure-like episodes, increased ataxia, andtransient hypotonia, as did animals that were given ibogainealone. However, in 3-AP-treated rats, ibogaine did not produce ahigh-frequency tremor, unlike normal rats that received ibogaine.After 15 min of hypotonia, there was a gradual return of truncaltone accompanied by ataxia, but the high-frequency tremor wasnot present. On the day after ibogaine treatment, these rats wereindistinguishable from those given the 3-AP and harmaline reg-imen alone; they displayed ataxia, mud-walking, and a brieftremor on initiation of movement. In summary, in rats treatedwith the 3-AP regimen, ibogaine produced myoclonus, seizure-like episodes, increased ataxia, and hypotonia, but no sustainedtremor, indicating that ablation of the inferior olive prevented thecharacteristic high-frequency tremor.

ImmunohistochemistryCerebellar cortex: control and ibogaine-treatedNormal, intact Purkinje cells are strongly immunoreactive withantibodies to calbindin or to Cam-kin II, which stain the cellbodies, axons, and dendrites. In untreated control rats, Purkinjecell bodies form an uninterrupted monolayer throughout thecerebellar cortex; the dendrites, which are densely stained, form anetwork of closely packed processes that extends continuouslythrough the molecular layer. In rats that were administered ibo-gaine alone, small groups of Purkinje cell bodies and dendriteswere selectively lost. At short survivals (1–2 d), irregular,shrunken, and fragmented Purkinje cell bodies were present (E.O’Hearn, unpublished observations). After a 1 week survival,Purkinje cell loss was manifest as multiple pale, radial bands thatwere unstained with neuronal markers (Fig. 1A,B). The neuronsbetween these pale bands remained intact and densely stained, asin control rats. Examination of a sequential series of coronalsections revealed clusters of degenerating or absent Purkinje cellsaligned in narrow parasagittal rows that typically ranged from oneto five Purkinje cells in width. These pale sagittal bands ofneuronal loss observed in cerebellar cortex 7 d after ibogainetreatment spanned the Purkinje cell and molecular layers (Fig.1A,B). Within these radial bands, Purkinje cell bodies and den-drites were not detectable using antibodies to calbindin or Cam-kin II. Adjacent Nissl-stained sections revealed small unstainedpatches in which Purkinje cell bodies were missing; these patcheswere in register with the gaps in calbindin and Cam-kin IIstaining seen in neighboring sections. Ibogaine-induced Purkinjecell loss was most prominent in the vermis of the cerebellum, butalso included degeneration in the paravermis and, much lesscommonly, in the hemispheres and paraflocculus.

Activated microglia were detected by intense staining withantibodies to the complement 3 receptor (OX42) (Fig. 1C,D) andto the MHC II antigen (OX6) (data not shown). Cell bodies andprocesses of activated microglia were enlarged and intenselyimmunoreactive compared with quiescent microglia located inadjacent zones (Fig. 1D). In control rats, activated microglia wereextremely uncommon, whereas the cerebellar cortex of ibogaine-treated rats exhibited clusters of large, darkly stained microglialcells located primarily in the molecular and Purkinje cell layers.Activated microglia formed distinct groups that were coextensivewith radial bands devoid of Purkinje cell bodies and dendrites, asseen in neighboring sections prepared for Cam-kin II (Fig. 1) orNissl stain. Both microglial markers (OX6 and OX42) stained thesame distribution of activated cells, but in controls the MHC IIantigen was expressed by resting microglia primarily in the cere-bellar white matter, whereas complement 3 receptor was ex-pressed in resting microglia throughout the cerebellum. In addi-tion, narrow stripes of activated astrocytes (Bergmann glia),densely stained with antibodies to GFAP, were observed in thePurkinje cell and molecular layers (data not shown). These clus-ters of activated astrocytes were distributed in register withdegenerating Purkinje cells and activated microglia.

Inferior oliveControl inferior olive. In the ventral medulla, neurons expressingcalbindin and Cam-kin II were densely packed in lamellae thatform the inferior olivary nucleus. Both neuronal markers had thesame distribution and appeared to stain all neurons in the inferiorolive. Multiple divisions of the inferior olive could be delineatedclearly in sections that were immunostained with antibodies tothese proteins. Nissl-stained sections revealed abundant oval-shaped neuronal cell bodies and nuclei. In untreated controlanimals, the inferior olivary nuclei showed no evidence of acti-vated microglia or astrocytes.

Effects of ibogaine alone on inferior olive. After ibogaine admin-istration, neurons of the inferior olivary nucleus, stained withanti-Cam-kin II (Fig. 2A,C), anticalbindin antibodies, or cresylviolet (Fig. 2E), were unchanged from controls and were normalin density and morphology. Within the inferior olivary nucleus,glial staining with OX42 (Fig. 2G) or GFAP antisera (data notshown) was not increased; inferior olivary microglia and astro-cytes were indistinguishable from quiescent glial cells found incontrol animals.

Effects of 3-AP regimen on inferior olive. After treatment withthe cytotoxic 3-AP regimen, nearly all neurons in the inferiorolive degenerated. In all 3-AP-treated rats, the region of themedulla containing the inferior olivary nucleus was largely de-pleted of neuronal cell bodies that were immunopositive foreither Cam-kin II (Fig. 2B,D) or calbindin. Nissl-stained sectionsrevealed that the site where the inferior olive is located containeddensely packed, small nuclei, presumably of glial cells (astrocytesand microglia) (Fig. 2F). Notably absent in these sections werethe large neuronal nuclei and somata that are present in sectionsfrom control (or ibogaine-treated) rats (Fig. 2E). After staining

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with pale zones in A). The largest and most activated microglia are located in the Purkinje cell layer, where they are presumably phagocytizing a Purkinjecell body (D). Resting microglia are the small, lightly stained cells with fine processes in C and D that are widely distributed throughout all layers ofcerebellar cortex and white matter. Microglia are immunoreactive with OX42, which recognizes the complement receptor 3B. Activated microglia aremore intensely immunoreactive and have larger processes and cell bodies ( D). M, Molecular layer; P, Purkinje cell layer; G, granule cell layer. Scale bars:A, C, 500 mm; B, D, 100 mm.


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Figure 2. Most neurons in the inferior olivary nucleus degenerate after administration of the 3-AP regimen used in this study. In animals treated withibogaine alone, inferior olive neurons remain intact and exhibit normal morphology (A, C); in contrast, profound loss of neurons is evident (B, D) in theinferior olive of rats that received the 3-AP regimen (3-acetylpyridine, harmaline, and nicotinamide; 13 d survival). A–D, Inferior olivary nucleus: Cam-kinII immunoreactivity at low (A, B) and high (C, D) magnification. E, Large neuronal cell bodies are shown with Nissl stain of (Figure legend continues)


with a microglial marker (OX42), sections through this regiondemonstrated that olivary neurons had been replaced by numer-ous, intensely activated microglial cells that were densely packedin a gliotic zone that resembled the inferior olivary nucleus inlocation and shape, as shown in Figure 2H.

Effects of 3-AP regimen, followed by ibogaine, on inferior olive. Inanimals that received the 3-AP regimen, followed 1 week later byibogaine, no further changes were detected in the inferior olive.Rats treated with the 3-AP regimen alone were compared withrats given 3-AP plus ibogaine. Sections of the inferior olive fromthe two groups were indistinguishable. As noted above, nearly allolivary neurons were ablated by the 3-AP regimen and werereplaced by activated glial cells. In both 3-AP treatment groups,

microglia and astrocytes in the inferior olivary nucleus wereconsistently enlarged and strongly immunoreactive to glial anti-bodies (GFAP, OX42, and OX6).

In 22 of 26 rats that received the 3-AP regimen, with or withoutibogaine, a small contingent of calbindin- or Cam-kin II-immunoreactive neurons remained in the inferior olive. Thesesurviving neurons, in small numbers, were found in particularsubdivisions of the olivary complex. Spared cells were typicallyconfined to the medial accessory olive and an occasional neuronsurvived within the dorsal accessory olive or the principal olive.

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Figure 3. Activated microglia exhibit a different distribution and morphology in ibogaine- versus 3-AP-treated animals. After ibogaine administration(100 mg/kg; 7 d survival), activated microglia form radial bands located primarily in the vermis (A); the most intensely activated microglia (C) are foundat the depth of Purkinje cell bodies (which have degenerated, as observed in adjacent sections). After the 3-AP regimen (13 d survival), occasionalactivated microglia are observed far laterally in the hemispheres (B, arrowhead), primarily in the ansiform lobule, crus I, and less commonly in the vermis;these activated cells are found mainly in the molecular layer and tended not to be adjacent to Purkinje cell bodies (D). Enlarged, darkly immunoreactivecells are activated microglia. More delicate cellular profiles (C, D) with finer processes, smaller cell bodies, and less intense immunostaining are quiescentmicroglia. In ibogaine-treated animals, activated microglia ( C) are larger and more darkly immunoreactive than those seen after the 3-AP regimen ( D),suggesting that 3-AP may cause a smaller degree of neuronal insult. Coronal sections immunostained with antibody OX42 for complement receptor 3B.Scale bars: A, B, 500 mm; C, D, 50 mm. P, Purkinje cell layer.

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rats ( E), whereas smaller profiles are glial cells. F, After the 3-AP regimen, neuronal profiles in the inferior olive are absent, and this nucleus has becomedensely populated with small glial cell bodies. The inferior olive is gliotic because of proliferation of astrocytes and microglia that were activated inresponse to degeneration of inferior olive neurons. Using a marker for microglial cells (G, H ), only lightly stained, resting microglia are observed in theinferior olive from an ibogaine-treated rat (G); in contrast, after the 3-AP regimen, densely packed activated microglia occupy the site of the formerinferior olivary nucleus ( H ) and demarcate the different subregions that were present in this nucleus. Cytochemical markers and stains: A–D, Cam-kinII immunocytochemistry for inferior olive neurons; E, F, Nissl stain of inferior olive; G, H, to identify microglia, the inferior olive is stained with antibody(OX42) that recognizes the complement receptor 3B. This receptor is expressed at moderate levels by quiescent microglia and is greatly increased inactivated microglia in response to neuronal injury or degeneration. Scale bars: A–H, 100 mm.


Cerebellar cortex af ter ablation of inferior oliveEffects of 3-AP regimen on cerebellar cortex . In the 3-AP-treatedrats, as in controls, Purkinje cell bodies formed a monolayerthroughout the cerebellar cortex. Neuronal morphology after3-AP could not be distinguished from that in control rats, as seenin sections stained for Nissl or for other neuronal markers. Themolecular layer, containing dendrites of Purkinje cells and otherneurons, was well stained with antisera to Cam-kin II and cal-bindin. However, a minor effect of the 3-AP regimen could bedetected on close examination. Two weeks after 3-AP treatment,there were signs suggesting that a small number of individualPurkinje cells were missing. Sections stained for Cam-kin IIoccasionally revealed a narrow unstained radial band across themolecular and Purkinje cell layers with a width equal to onePurkinje cell. Such bands were infrequent and appeared to belocated primarily in the lateral portion of the hemispheres.

Resting microglia, immunostained for OX42, were observedthroughout cerebellar cortex in both 3-AP-treated and controlrats. After receiving 3-AP, a thin radial column of mildly acti-vated microglia was occasionally found in the molecular layer(Fig. 3B,D) but generally did not extend into the Purkinje celllayer (Fig. 3D). Columns of activated microglia were infrequentand nearly always one cell in width, when present. Thin microglialcolumns seen after 3-AP were predominantly located not in thevermis, but in the lateral part of the cerebellar hemisphere,mainly in crus I of the ansiform lobule (Fig. 3B,D). These micro-glia were darker than resting microglia but typically were lessintensely stained or enlarged than the highly activated microgliaseen after ibogaine (Figs. 1C,D, 3A,C). The cytotoxic effects ofthe 3-AP regimen in the cerebellum were subtle and differedmarkedly from the effects of ibogaine. In rats that receivedibogaine alone, dense columns of activated microglia were foundprimarily in the vermis (Figs. 1C, 3A) and in the medial part ofthe simple lobule, unlike the more lateral distribution seen in3-AP-treated rats (Fig. 3B,D). Moreover, the largest and mostintensely activated microglial cells after ibogaine were located atthe level of Purkinje cell bodies (Figs. 1D, 3C); in contrast,3-AP-induced glial activation did not usually extend down to thePurkinje cell layer (Fig. 3D). In addition, after 3-AP treatmentthere were occasional thin radial stripes of increased GFAP in themolecular layer (data not shown). The preceding results suggestthat the 3-AP regimen alone may have caused loss of an occa-sional neuron (Purkinje, stellate, or basket cell), yet neuronaldamage attributed to the 3-AP regimen was orders of magnitudeless than that produced by ibogaine. In addition, the distributionof neuronal cell loss and glial activation differed between animalsthat received ibogaine versus the 3-AP regimen. In summary,neuronal damage after the 3-AP regimen was typically found inlateral parts of the hemispheres rather than in the vermis (Fig.3B,D), in contrast to degeneration induced by ibogaine, which waslocated predominantly in the vermis (Figs. 1, 3A). At the 2 weeksurvival time, when 3-AP-treated animals were studied, there wasno evidence of widespread microglial activation that could beascribed to loss of climbing fibers.

Effects of 3-AP regimen, followed by ibogaine, on cerebellarcortex . Ablation of the inferior olivary nucleus with the 3-APregimen markedly attenuated Purkinje cell degeneration causedby subsequent ibogaine treatment (Figs. 4, 5). Rats that receivedibogaine alone were compared with those that received the 3-APregimen followed by ibogaine 6 d later. In both experimentalgroups, the survival time after ibogaine administration was 1

week. After administration of 3-AP before ibogaine, Purkinje cellloss was markedly reduced (Fig. 4); in contrast to the prominentbands of neuronal loss that followed treatment with ibogainealone (Fig. 4A,C,E), zones of missing Purkinje cells were nearlyabsent in the vermis of animals that received 3-AP plus ibogaine(Fig. 4B,D,F). This neuroprotective effect of olive ablation wasseen in 17 of 17 animals that were treated with the 3-AP regimenbefore ibogaine. Yet, protection against Purkinje cell degenera-tion in these rats was not absolute, because a thin band ofneuronal degeneration was occasionally found in the vermis orhemisphere. The minimal neurotoxicity found after 3-AP plusibogaine was detected by the presence of infrequent narrow bandsunstained with neuronal markers, located predominantly in thelateral part of the hemisphere. This distribution of cell loss in thelateral hemisphere is similar to the loss observed in animals thatreceived the 3-AP regimen by itself. In contrast, rats that receivedibogaine alone exhibited numerous radial gaps in staining, mainlyin the vermis where Purkinje cells had degenerated, as seen incalbindin, Cam-kin II (Figs. 1A,B, 4A,C,E), and Nissl sections.

The distribution of activated microglia and astrocytes paral-leled that of neuronal loss. Compared with the effects of ibogainealone, microglial activation was markedly decreased in rats thatreceived the 3-AP regimen followed by ibogaine (Fig. 5B,D,F).These animals exhibited only occasional radial stripes of activatedmicroglia; the few glial stripes present were narrow and werelocated either in the vermis or in the lateral part of the hemi-sphere. The lateral microglial stripes were primarily situated inthe ansiform lobule, the same location where activated microgliawere also found after the 3-AP regimen alone (Fig. 3B,D). Theseresults show that previous ablation of the inferior olive by 3-APalmost completely prevented ibogaine-induced degeneration ofPurkinje cells and activation of neighboring glial cells. The lateralposition of residual neuronal damage observed after 3-AP plusibogaine may result from toxicity caused by the 3-AP regimenitself, before administration of ibogaine.

3-AP toxicity in other brain sitesIn agreement with previous studies, evidence suggesting minor3-AP-induced neuronal injury was seen in regions outside of thecerebellar cortex. After the 3-AP treatment regimen, moderatelyincreased OX42 staining indicative of activated microglia wasfound in the deep cerebellar nuclei in addition to other brainstemnuclei, which included the lateral vestibular, dorsal cochlear,ambiguus, and hypoglossal nuclei (data not shown). 3-AP-induced activation of microglial cells in the deep cerebellar nuclei(fastigial, interposed, and dentate) produced an equivalent de-gree of enhanced microglial staining across these three nuclei.This distribution of activated glial cells differed from that ob-served after ibogaine alone, in which the fastigial nucleus con-tained significantly more prominent microglial activation than didthe interposed or dentate nuclei (data not shown). In ibogaine-treated rats there were no neuronal changes in Nissl-stainedsections of the deep cerebellar nuclei, suggesting that microglialactivation in these nuclei resulted from degeneration of axonterminals, quite likely those arising from Purkinje cells. Gliosispredominantly in the fastigial nucleus is consistent with degener-ation of Purkinje cells in the vermis, the main location ofneurotoxicity.

DISCUSSIONSystemic administration of ibogaine in rats produces cerebellarneurotoxicity manifested by the selective loss of a small population


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3-AP regimen + IbogaineIbogaineFigure 4. A–F, Neuroprotection: ablation of the inferior olive with 3-AP prevents or greatly attenuates Purkinje cell degeneration induced by ibogaine.Left column (A, C, E), Treatment with ibogaine alone produces radial bands of Purkinje cell loss manifested by pale, unstained gaps in the molecularand Purkinje cell layers. Loss of Purkinje cells is most prominent in the vermis but is also present in the paravermis and simplex lobule. Right column(B, D, F ), Animals that received the 3-AP regimen followed by ibogaine 6 d later demonstrate marked neuroprotection against Purkinje celldegeneration. The nearly continuous immunostaining of Purkinje cell bodies and of their dendrites in the molecular layer (B, D, F ) indicates that thereis little or no ibogaine-induced degeneration of Purkinje cells after olive ablation. Infrequently in rats that received the 3-AP regimen plus ibogaine, asingle Purkinje cell may have degenerated (see thin gap in neuronal staining of molecular and Purkinje cell layers in upper right corner of D).Photomicrographs show Purkinje cells in coronal sections immunostained with antiserum to Cam-kin II. Ibogaine dose, 100 mg/kg once; in all cases,survival was 7 d after ibogaine administration. Scale bars: A, B, 500 mm; C–F, 100 mm.


A B

C D

E F

Ibogaine 3-AP regimen + Ibogaine

Figure 5. Ablation of the inferior olive with the 3-AP regimen profoundly attenuates subsequent microglial activation induced by ibogaine. Left column(A, C, E), Treatment with ibogaine alone produces radial bands of darkly stained, activated microglia, primarily in the molecular and Purkinje cell layersof the cerebellar vermis. In most cases, the radial bands of microglia are coextensive with bands of degenerating Purkinje cells (Fig. 1). Right column (B,D, F ), Animals that received the 3-AP regimen followed by ibogaine 6 d later demonstrate few signs of microglial activation. After the 3-AP regimen,ibogaine no longer produces more than an occasional radial stripe of activated microglia. The great majority of cells seen are resting microglia that arefaintly stained and have delicate processes. Activated microglia are infrequent in the rats that received the 3-AP regimen plus ibogaine. Photomicro-graphs of coronal sections show microglial cells immunostained with antiserum OX42 for complement receptor 3B. Drug doses: A, C, E, ibogaine (100mg/kg); B, D, F, 3-AP regimen given 6 d before ibogaine (100 mg/kg). In all cases, survival was 7 d after ibogaine administration. Scale bars: A, B, 500mm; C, D, 100 mm; E, F, 50 mm.


of Purkinje cells (O’Hearn and Molliver, 1993). Purkinje cells thatundergo degeneration are distributed in discrete longitudinal rowsthat are coextensive with bands of activated microglia and astro-cytes (O’Hearn et al., 1993). These multiple bands of neuronaldegeneration, which form radial stripes within the molecular andPurkinje cell layers, are found predominantly in the vermis butinfrequently in the paravermis and cerebellar hemispheres(O’Hearn and Molliver, 1993; Molinari et al., 1996). The main newfinding of this investigation is that ablation of the inferior olivarynucleus by 3-AP prevents subsequent ibogaine-induced Purkinjecell degeneration. This result leads to the conclusion that ibogaineis not directly toxic to Purkinje cells but instead causes Purkinjecell degeneration through sustained activation of the olivocerebel-lar projection. This paper presents evidence that ibogaine-inducedPurkinje cell degeneration provides an experimental in vivo para-digm of trans-synaptic, excitotoxic neuronal degeneration, which ismediated through intrinsic olivocerebellar circuitry.

The spatial pattern of Purkinje cell loss induced by ibogainesupports a primary role for the olivocerebellar projection inproducing this form of neurotoxicity. The longitudinal band-likedistribution of Purkinje cell degeneration caused by ibogainecorresponds to the sagittal organization of inferior olive–Pur-kinje cell innervation. Earlier studies revealed that the inferiorolive projects topographically to longitudinal zones of cerebellarcortex (Oscarsson, 1976) such that small clusters of olivary neu-rons innervate Purkinje cells that are aligned in parasagittal rowsthat are hundreds of micrometers wide (Groenewegen andVoogd, 1977; Brodal and Kawamura, 1980; Azizi and Woodward,1987). By recording from multiple Purkinje cells simultaneously,Llinas and colleagues elegantly demonstrated that climbing fiberactivation occurs synchronously in Purkinje cells that lie in rostro-caudal rows (Llinas and Sasaki, 1989; Sasaki et al., 1989). Theselongitudinally oriented, physiological “microzones” are narrowerthan the zones defined anatomically, encompassing from one toseveral Purkinje cells. Moreover, clusters of inferior olivary neu-rons are electrotonically coupled by gap junctions (Llinas et al.,1974; Sotelo et al., 1974; Llinas and Yarom, 1986) and synchro-nously excite Purkinje cells in the same longitudinal row (Llinasand Sasaki, 1989; Sasaki et al., 1989).

We postulate that the principal effect of ibogaine, like therelated compounds harmaline and ibogaline (Fig. 6), is to in-crease the activity of neurons in the inferior olive. These threedrugs have similar behavioral and pharmacological effects, and allof them produce a marked tremor. Several lines of evidencesuggest that the tremor induced by these indole alkaloids resultsfrom excitation of inferior olivary neurons. The most thoroughlycharacterized of these drugs is harmaline, which causes a sus-tained 8–12 Hz generalized tremor in all species tested, includingmice (Zetler, 1957), rats (Zetler et al., 1972, 1974), cats (Lamarreet al., 1971), and monkeys (Battista et al., 1990). Ibogaline andibogaine produce an identical 8–12 Hz tremor in mice (Zetler etal., 1972) and in cats (De Montigny and Lamarre, 1974). Inaddition, harmaline induces sustained rhythmic activation of in-ferior olivary neurons when given systemically (Lamarre et al.,1971; De Montigny and Lamarre, 1973; Llinas and Volkind,1973), when microinjected directly onto inferior olive neurons invivo (De Montigny and Lamarre, 1975), or when applied bysuperfusion of the inferior olive in slices (Llinas and Yarom,1986). Harmaline-treated animals demonstrate synchronous,rhythmic bursts of activity (8–12 Hz) throughout the spino–cerebellar system, including inferior olive, Purkinje cells, deepcerebellar nuclei, reticular formation, and motoneurons (Lamarre

and Mercier, 1971; Lamarre et al., 1971; Lamarre and Weiss,1973; Llinas and Volkind, 1973). Bursts of rhythmic neuronalactivity are time-locked to the harmaline-induced tremor, asassessed by electromyographic recording (De Montigny andLamarre, 1973; Milner et al., 1995). These data indicate that thetremor is induced by rhythmic activation of motoneurons attrib-utable to entrainment of brainstem descending pathways by theolivocerebellar rhythm. Initial proposals that the harmalinetremor originates in inferior olivary neurons have received con-siderable experimental support. Lesions that interrupt connec-tions between the inferior olive and cerebellum prevent theharmaline tremor but do not block drug-induced activation ofinferior olivary neurons (Lamarre and Mercier, 1971; De Mon-tigny and Lamarre, 1973; Llinas and Volkind, 1973). Chemicalablation of the inferior olive by 3-AP also prevents harmaline-induced tremor (Simantov et al., 1976).

This laboratory reported previously that both ibogaine andharmaline have similar behavioral effects and produce the samepattern of Purkinje cell degeneration (O’Hearn and Molliver,1993). Based on their structural similarities (Fig. 6) and identicalbehavioral and neurotoxic effects, it is likely that ibogaine, likeharmaline, acts directly on inferior olivary neurons to initiatesynchronous rhythmic activity. The present results show thatolive ablation prevents ibogaine-induced tremor, supporting theproposal that ibogaine acts on the inferior olive. However, thefinding that ibogaine produces hypotonia despite olive ablationsuggests that the hypotonia is not mediated by the olivocerebellarprojection, and that this drug acts at additional CNS sites. Othersites of ibogaine action were suggested by extensive c-Fos induc-tion caused by ibogaine given after inferior olive ablation(O’Hearn and Molliver, 1994).

Figure 6. Chemical structures of the indole alkaloids harmaline, ibo-gaine, and ibogaline. Harmaline, ibogaine, and ibogaline share an indolenucleus and have nearly identical types of behavioral effects. Zetler et al.(1972, 1974) reported that the positions and number of methoxy groups(lef t) greatly influence tremorigenic potency, whereas the additional ringstructures (right) have little effect on drug action within this group ofcompounds. For comparative potencies of tremor induction, see Zetler etal. (1972, 1974).


Although harmaline and related drugs clearly produce activationof olivary neurons, particular subdivisions of the inferior olivarycomplex differ in their susceptibility to this effect. This regionalselectivity may partially explain the localization of ibogaine toxicityto the vermis. Both harmaline and ibogaline activate neuronspreferentially in caudal portions of the medial and dorsal accessoryolivary subnuclei (De Montigny and Lamarre, 1973, 1974; Llinasand Volkind, 1973; Batini et al., 1981), regions of the inferior olivethat project selectively on the vermis (Groenewegen and Voogd,1977; Brodal and Kawamura, 1980; Azizi and Woodward, 1987;Buisseret-Delmas and Angaut, 1993). Moreover, dendro-dendriticgap junctions, which likely contribute to rhythmic activation, areprimarily located in a restricted portion of the medial inferior olive(de Zeeuw et al., 1996). The specific mechanism by which this classof compounds increases excitability and inferior olive firing (from1–2 to 8–12 Hz) is not fully established, yet Llinas and Yarom(1986) have demonstrated that harmaline leads to hyperpolariza-tion of olivary neurons and facilitates calcium conductance throughlow threshold calcium channels.

The present results suggest that the distribution of climbingfibers in sagittal bands determines the spatial pattern of degen-eration induced by ibogaine. Moreover, the uniquely dense syn-aptic relationship of climbing fibers with Purkinje cells is likely tounderlie the potential for neuronal injury in this system. Climbingfibers arise exclusively from inferior olivary neurons (Szen-tagothai and Rajkovits, 1959; Desclin, 1974), and each Purkinjecell is innervated by a single climbing fiber from the contralateralinferior olive. Climbing fiber axons pass over the Purkinje cellbody and form branches that closely follow the proximal two-thirds of Purkinje cell dendrites (Eccles et al., 1967; Palay andChan-Palay, 1974). Each climbing fiber forms several hundredsynaptic contacts on dendritic spines distributed over one Pur-kinje cell (Llinas et al., 1969), resulting in a unique and extensiveexcitatory synaptic input. This “distributed” synaptic arrange-ment provides a structural basis for the powerful excitatory actionof climbing fibers on Purkinje cells (Eccles et al., 1966a,b). Everyclimbing fiber action potential results in nearly synchronous re-lease of glutamate, the most likely transmitter of olivary neurons(Zhang et al., 1990), at hundreds of synapses distributed over thesurface of the Purkinje cell dendritic tree. The release of gluta-mate simultaneously at every synaptic terminal on a Purkinje cellproduces “complex spikes,” consisting of an initial spike followedby a high-frequency burst of smaller calcium-mediated actionpotentials (Eccles et al., 1966a,b; Llinas and Nicholson, 1971;Llinas and Sugimori, 1980). The multiplicity of climbing fibersynapses is responsible for the highly secure synaptic transmissionin this projection, yet this synaptic relationship also places thePurkinje cell in substantial jeopardy of excitotoxic injury.

Based on the hypothesis that ibogaine, like harmaline andibogaline, causes a sustained increase in frequency of neuronalfiring in the inferior olive, we propose that this property, com-bined with the highly secure synaptic relationship between climb-ing fibers and Purkinje cells, results in Purkinje cell injury by anexcitotoxic mechanism. We further postulate that the distributednature of climbing fiber–Purkinje cell innervation renders Pur-kinje cells especially susceptible to excitotoxic injury and may bethe basis for the heightened vulnerability of Purkinje cells to awide variety of insults. The incidence of Purkinje cell loss inhumans is disproportionate to the loss of other neurons afterhypoxia or ischemia, prolonged seizures, exposure to numeroustoxins, in normal aging, and in several neurodegenerative disor-ders (Blackwood and Corsellis, 1976; Adams and Duchen, 1992).

Excitotoxic neuronal injury is proposed to result from intense andprolonged glutamatergic excitation that produces excessive eleva-tion of intracellular calcium, leading to activation of multiplecalcium-dependent enzymes that damage cellular constituents(Olney et al., 1971; Olney, 1978; Garthwaite et al., 1986; Choi,1987, 1988; Siman and Noszek, 1988). Activation of Purkinje cellsby climbing fibers can lead to elevation of cytosolic calcium levelsby several mechanisms. Glutamate receptors that are postsynapticto climbing fiber terminals are of the non-NMDA (AMPA orkainate) (Konnerth et al., 1990; Perkel et al., 1990), or metabo-tropic receptor subtype (Masu et al., 1991; Martin et al., 1992;Baude et al., 1993). Although NMDA receptors are expressedtransiently by Purkinje cells during early development, in matureanimals Purkinje cells do not exhibit functional NMDA receptors(Crepel et al., 1982; Perkel et al., 1990; Crepel and Audinat, 1991;Rosenmund et al., 1992), and they would therefore not play a rolein neurotoxicity attributed to ibogaine or harmaline.

Glutamate activation of AMPA receptors on Purkinje cellsproduces influx of sodium, leading to membrane depolarization,secondarily allowing influx of extracellular calcium throughvoltage-sensitive calcium channels (Kostyuk, 1990; Bertolino andLlinas, 1992; Miyakawa et al., 1992). Additionally, a subset ofAMPA receptors is capable of transmitting calcium directly, inaddition to sodium and potassium (Sorimachi, 1993; Brorson et al.,1994; Jonas et al., 1994). Stimulation of metabotropic receptors onPurkinje cells leads to the formation of inositol 1,4,5-trisphosphate,which produces release of calcium from storage vesicles in endo-plasmic reticulum (Ross et al., 1989; Satoh et al., 1990; Llano et al.,1991). Thus, repetitive and prolonged climbing fiber activation,through the combination of several postsynaptic mechanisms, iscapable of producing massive increases in levels of intracellularcalcium throughout the Purkinje cell cytoplasm, in both soma anddendrites. These sustained high concentrations of cytosolic calciumare capable of activating intracellular enzymes, such as lipases andproteases, that may initiate a proteolytic cascade leading to Pur-kinje cell degeneration (Brorson et al., 1994; Orrenius et al., 1996).

Other InterpretationsAlthough the most likely interpretation of the present results isthat ibogaine-induced Purkinje cell degeneration is trans-synaptically mediated via the olivocerebellar projection, the pos-sible involvement of other mechanisms and factors should beconsidered. (1) Ibogaine might have a direct, deleterious effect onPurkinje cells that could impair their response to excessive exci-tatory input and, combined with increased climbing fiber activa-tion, may contribute to degeneration. Sparing of Purkinje cellsand the preservation of their normal morphology in rats thatreceived ibogaine after ablation of the inferior olive does not lendsupport to this interpretation. Moreover, it is unlikely that ibo-gaine has direct neurotoxic effects, because it did not producesigns of neuronal injury in the inferior olive, a region in which thedrug has significant physiological effects. However, it is difficult toexclude the possibility that ibogaine may have an undetectedneurotoxic effect that produces no morphological or immunocy-tochemical changes. (2) An alternative explanation for these datais that, in addition to sustained climbing fiber activation becauseof ibogaine, the presence of severe tremor might indirectly lead toincreased activity in parallel fibers. The combination of increasedclimbing and parallel fiber activity together may contribute toPurkinje cell injury. Two factors make this explanation unlikely:(A) harmaline causes sustained rhythmic bursts of complex spikesin Purkinje cells, during which “simple spikes,” the signs of


parallel fiber synaptic input, are suppressed (Lamarre et al.,1971); and (B) parallel fibers traverse the folium in a mediolateraldirection, at right angles to the rows of degenerating Purkinjecells, whereas the sagittal bands of degeneration match the dis-tribution of climbing fibers. (3) A third caveat arises from reportsthat neuronal injury after 3-AP may not be restricted to inferiorolive neurons. Several neuronal groups are reported to degener-ate after treatment with 3-AP, including nucleus ambiguus, hy-poglossal nuclei, dorsal motor nucleus of vagus, interpeduncularnuclei, substantia nigra, ventral tegmental area, and dentate gyrus(Balaban, 1985). If Purkinje cell degeneration was increased byactivity in these nuclei, then previous administration of 3-APmight theoretically attenuate the neurotoxic effects of ibogaine onPurkinje cells. However, none of the other neuronal groups thatmay be injured by 3-AP is known to innervate Purkinje cellsdirectly. Moreover, the 3-AP regimen used in this study, whichincludes a small dose of harmaline followed by nicotinamide, isthought to maximize the selectivity of degeneration in the infe-rior olive (Llinas et al., 1975). Although these alternative expla-nations cannot be completely excluded, the most parsimoniousinterpretation of the present results is that ibogaine causes exci-totoxic Purkinje cell degeneration mediated via the olivocerebel-lar projection.

Sparing of inferior olive neuronsAlthough ablation of the inferior olive with 3-AP affords substan-tial protection against Purkinje cell degeneration, the attenuationis not complete, because loss of an occasional Purkinje cell wasobserved in some rats that received the 3-AP regimen beforeibogaine. Relevant to this finding is that not all neurons in theinferior olive degenerated in every animal that received the 3-APregimen. The relatively few inferior olivary neurons that survived3-AP treatment were most commonly located within the medialaccessory olive. Differential susceptibility of inferior olivary neu-rons to the 3-AP regimen has been reported previously, withsurviving neurons found most frequently in caudal portions of theinferior olive, especially in the medial accessory olive (Llinas etal., 1975; Anderson and Flumerfelt, 1984; Rossi et al., 1991),which is known to project to the vermis (Brodal, 1954). Thepersistence of these olivary neurons, along with their climbingfiber projections to the cerebellum, might contribute to the infre-quent ibogaine-induced degeneration of Purkinje cells that wasobserved in the vermis of some 3-AP-treated animals.

Relevant to interpreting sporadic Purkinje cell degenerationafter ablation of the olive is the finding that a small number ofPurkinje cells degenerate after treatment with the 3-AP regimenalone, a result not reported previously. After the 3-AP regimen,occasional narrow gaps in Purkinje cell staining in the vermis andansiform lobule (crus I) were coupled with similarly infrequentradial bands of activated microglia, a combination suggesting Pur-kinje cell damage. Compared with degeneration caused by ibo-gaine, Purkinje cell loss resulting from the 3-AP regimen alone isconsiderably less, and neuronal degeneration produced by thesetwo drugs is dissimilar in distribution. After the 3-AP regimen,Purkinje cell damage is located mainly in the lateral part of thehemispheres, whereas ibogaine induces neuronal death primarilyin the vermis. After receiving 3-AP plus ibogaine, Purkinje cellloss, although infrequent, was slightly more common in the vermisthan after 3-AP alone; however, no increase in degeneration wasappreciated in the lateral part of the hemispheres compared withthat caused by 3-AP alone. Based on this combination of results,

much of the Purkinje cell loss after treatment with 3-AP plusibogaine likely results from cytotoxicity of the 3-AP regimen itself,which includes a low dose of harmaline. Alternatively, the addi-tional Purkinje cell degeneration in the vermis may be because ofibogaine-induced activation of spared inferior olivary neurons.

Ablation of the inferior olivary nucleus with subsequent degen-eration of climbing fibers may result in altered Purkinje cellactivity and morphology that should be considered in interpretingthe present data. The removal of climbing fibers leads to Purkinjecell changes that might possibly render those cells less sensitive tocytotoxic injury. Within minutes after climbing fiber deafferen-tation, Purkinje cells double the frequency of simple spikes, andthe firing pattern becomes significantly more regular comparedwith Purkinje cells with intact climbing fibers (Colin et al., 1980).The increase in simple spike activity persists for several weeksafter olive ablation (Benedetti et al., 1984). Six days after the3-AP regimen, the time point when ibogaine was administered inthis study, deafferented Purkinje cells would likely be firing sim-ple spikes at rates well above normal. It is unlikely that theincreased firing rate would protect Purkinje cells against the toxiceffects of ibogaine.

In addition to increased activity after climbing fiber abla-tion, deafferented Purkinje cells undergo morphologicalchanges. C limbing fibers are required for maintenance ofnormal Purkinje cell dendritic structure (Sotelo et al., 1975;Bradley and Berry, 1976). C limbing fiber deafferentation in-duced by 3-AP occurs within 18 –24 hr (Desclin and Escubi,1974; Sotelo et al., 1975) and is followed by an increase in thedensity of spines on secondary and tertiary dendritic branchesof the Purkinje cells. The increase in dendritic spines wouldnot predictably diminish excitotoxic vulnerability of thesecells. Possible axonal regeneration may also affect the neuro-toxicity. Partial inferior olive ablation is followed by sproutingof new collaterals that reinnervate neighboring Purkinje cells(Rossi et al., 1991). Although reinnervation might be expectedto favor Purkinje cell degeneration mediated by survivingclimbing fibers, the sprouting takes longer than 6 d and wouldnot likely be functional at the time of ibogaine administrationin this study. Although climbing fiber deafferentation is fol-lowed by changes in Purkinje cell structure and function, noneof these is reasonably expected to be neuroprotective to Pur-kinje cells. Taken together, the present results demonstratethat Purkinje cell degeneration attributed to ibogaine is indi-rect, mediated by the olivocerebellar projection. This exampleof neurotoxicity, using intrinsic circuitry that uses glutamate asneurotransmitter, supports the hypothesis that ibogaine causestrans-synaptic, excitotoxic neuronal degeneration.

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